Typically, particles including molecules, beads, microorganisms and cells are detected and quantified directly by microscopic, nephelometric or electronic enumeration, or indirectly, by the measurement of microorganism or cell metabolic activity, the use of chromogenic or fluorogenic dyes, or the incorporation of radioactive precursors (e.g., a compound containing a radioactive metal isotope). When employed for detection and quantification, the chromogenic or fluorogenic dyes and the radioactive precursors are attached to the particles to be detected and quantified. As such, the chromogenic or fluorogenic dyes and the radioactive precursors act as labels for the detection of the target particle.
However, labeling of particles by means of chromogenic or fluorogenic dyes and radioactive precursors often requires intermediate bridging molecules or functional groups, or metabolic processing to couple the target particle to the label. These methods, employing a bridging agent, are labor intensive and often require long incubation and processing times.
The use of porphyrin (or porphin) and its many derivatives as labels for various assays is known in the art. Porphyrins are tetrapyrrolic pigment macrocycles which may be found free in nature but which more commonly occur as complexes with metal ions, typically divalent metal ions. The base porphyrin structure is indicated in Formula 1. The numbers (1-8) indicate the positions at which groups can be attached to form the different porphyrin derivatives. ##STR1##
In various derivatives, individual groups may be present at each of the numbered substitution points. Examples of such derivatives include aetioporphyrin which exists as four different isomers in which the beta hydrogens of each pyrrole group have been replaced with a methyl and an ethyl group. Uroporphyrin is similar except that acetic acid and propionic acid groups are used instead of methyl and ethyl groups. Coproporphyrins similarly contain four methyl and four propionic acid groups. A final exemplary group are the protoporphyrins, a group of fifteen isomers having four methyl groups, two vinyl groups, and two propionic acid groups at positions 1-8. Shown below as Formula 2 is protoporphyrin IX. ##STR2##
In other derivatives, a single substitution may cause both of the numbered positions of each pyrrole group to be occupied. Phthalocyanine is typical of the porphyrin derivatives of this type. Phthalocyanine is also characterized by the presence of four nitrogen atoms which provide the link between the four pyrrole groups of the porphyrin nucleus. In the original porphyrin and the derivatives previously described, this linkage was provided by carbon atoms having a single terminal hydrogen atom bonded to each carbon. Shown below as Formula 3 is phthalocyanine. ##STR3##
As previously indicated, porphyrin and its various derivatives are typically complexed with metals. When this occurs, the two hydrogen atoms bonded to the nitrogen atoms of two diagonally opposed pyrrole groups are replaced by a single metal atom, M, as indicated in Formula 4. ##STR4##
Typical metals, M, which can be incorporated into the porphyrin structure are Iron (Fe), Cobalt (Co), Gallium (Ga), Tin (Sn), Zinc (Zn), Chromium (Cr), Magnesium (Mg), and the various elements of the lanthanide series. The various derivatives of the base porphyrin can be similarly complexed with a metal (M).
Porphyrin and its derivatives (hereafter referred to as porphyrin) have been successfully employed as labels (or markers) in immunoassays, nucleic acid probe assays, immunoblotting, hybridization assays, microscopy, imaging, flow cytometry, DNA sequencing, and photodynamic therapy. However, the use of porphyrin and its derivatives as a label for the chemiluminometric, radiometric, or fluorometric detection of a particle in these various assays and techniques has heretofore required that a bridging agent be used. In the art, the use of a bridging agent may be indicated by various terms, including but not limited to bridged(ing), coupled(ing), conjugated, linkage, and tethered. The bridging agent whether it is a molecule reactant or functional group couples the porphyrin label to the particle to be detected and quantified. The choice of porphyrin and bridging agent will vary significantly depending upon numerous criteria including the nature of the target particle, the media in which the target particle is located, and the means of detection to be employed. The bridging agent providing the linkage can be substituted onto the porphyrin at any number of possible positions, including for example one of the beta positions of a pyrrole group, a terminal position on some group substituted at the pyrrole group (e.g., replacing a hydrogen bound to a carbon of the six-member ring characteristic of phthalocyanine), or a terminal position at the carbon linking the pyrrole groups. The bridging agent may be substituted on the porphyrin at a single position or optionally it may be substituted at multiple positions. Positions at which the bridging device has been substituted onto the porphyrin but at which coupling does not occur may provide increased solubility. Indeed, substituents may be provided on to the porphyrin for the sole purpose of increasing solubility.
Numerous bridging agents (i.e., functional groups) are known in the art. They include but are not limited to sulfonic acid groups (--SO.sub.3 H), sulfonate groups (--SO.sub.3.sup.-, X.sup.+), carboxylic acid groups (--CO.sub.2 H), carboxylate groups (--CO.sub.3 2.sup.-, X.sup.+), phosphoric acid groups (--PO.sub.4 H.sub.2), phosphate groups (--PO.sub.4.sup.-, 2X.sup.+), phosphonate groups (--PO.sub.3.sup.-, X.sup.+ or --PO.sub.3 H), hydroxy or phenoxy groups (--OH), amino groups (--NH.sub.2) and ammonium and pyridinium groups (--NR.sub.4.sup.+, X.sup.-). Additionally, one of the most common ways to provide linkage is to form a water soluble carbodiimide derivative of the porphyrin. Optionally, the bridging agent can be coupled to the target particle first prior to its exposure to the porphyrin label.
In other detection methods, such as nucleic acid probe assays, it may be necessary to bind the porphyrin label to a nucleic acid primer or probe. A bridging agent, which may be initially bound to either the porphyrin label, the primer or probe, or the target particle, is used to effect the coupling of the porphyrin to the primer or probe. The primer or probe subsequently attaches itself to the target particle. There is a need for a detection process utilizing porphyrin labels that does not require a bridging agent for the binding of the porphyrin label to the target particle.